Four Common Protein Interaction Techniques You Must Know

As the main bearer of life activities, the function of proteins has always been a star that has attracted much attention in scientific research activities. Proteins are usually not "fighting alone", and the vast majority of functional proteins will interact with other proteins to regulate life processes together. So, how much do you know about the technology of protein interaction?

I. Co-immunoprecipitation

Co-immunoprecipitation (COIP) technique is based on the principle of specific immune binding between antigen and antibody. Specific antibody is added to cell lysis to precipitate antigen and antigen-bound protein. Immune complexes can verify the interaction between antigens and other proteins by Western blot, and can also detect the binding protein members of antigens by mass spectrometry.

Advantages and disadvantages of COIP technology:

Advantages

1. The obtained interacting proteins are naturally interacting with bait proteins in cells, which are in line with the real physiological situation in vivo; (2) The experimental conditions are mild and artificial effects can be avoided; (3) The interacting protein complexes in the native state can be isolated.

Disadvantages:

1. It is not applicable to the study of protein interaction with weak or instantaneous binding.

II. Pull Down

Pull-down techniques use solid-phase, labeled bait proteins or tag proteins (biotin-, PolyHis-, or GST-) to fish out proteins that interact with them from cell lysis. The interaction relationship between known proteins and phishing proteins or purified related proteins can be determined by pull-down technology, and the protein interaction relationship can be detected from in vitro transmission or translation systems. The protein interaction detected by COIP may be established by the third protein as a bridge, in contrast to the pull down technique, which can be used to detect direct interaction between proteins. However, pull down requires prokaryotic expression and purification of the bait protein, followed by incubation with the target protein solution, which cannot mimic the natural interaction environment in cells like COIP.

III. Bimolecular fluorescence complementation

Bimolecular fluorescence complementation technique refers to the incision of fluorescent protein polypeptide chains at some unconserved amino acids to form two non-fluorescent N- and C-terminal polypeptide fragments. These two fluorescent protein fragments were ligated to a pair of target proteins that could interact, respectively, and when these two fusion proteins were co-expressed intracellularly or mixed in vitro, due to the interaction of the target proteins, the two fragments of the fluorescent proteins were spatially close to each other and complementary to each other, and a complete active fluorescent protein molecule was reconstructed, thus producing fluorescence. Visualization is the greatest feature of BiFC technology.

IV. Yeast two-hybridYeast two-hybrid system is an important method widely used in protein interactomics. The principle is that when the target protein and the bait protein specifically bind, the bait protein binds to the promoter of the reporter gene and initiates the expression of the reporter gene in yeast cells, and if the expression product of the reporter gene is detected, it indicates that there is an interaction between the two, otherwise there is no interaction between the two. In practice, people have developed one-hybrid system, three-hybrid system and reverse hybridization system according to needs.

In addition, the methods to study protein interaction include phage display technology, plasmon resonance technology, antibody and protein array technology.

Collected by Profacgen

We have established in our lab a series of assays to help with your research, including high throughput interaction screening assays such as Yeast two-hybrid screening and phage display technology, interaction strength and kinetics assays such as Surface Plasmon Resonance (SPR).

Collected by Profacgen, an insect cell protein production provider.